Laser-induced fluorescence spectroscopy has been employed to probe complex biological systems, like cells and tissues, for biochemical and morphological alterations associated with early evidence of diseases, potentially leading to non-invasive diagnosis in vivo. Once diagnosed, such tissue may be treated at their earliest stages, reducing or preventing the risk of developing clinically apparent diseases that often have associated morbidity or mortality, like cancer.
Steady-state fluorescence spectroscopy has been explored as a non-invasive means of detecting both cancers and pre-neoplasia (pre-malignancy) in vivo. In tissue fluorescence spectroscopy, light is used to probe endogenous biological fluorophores like collagen, elastin, NADPH, and tryptophan. Because tissue is an inhomogeneous medium, fluorescence signals measured in vivo reflect tissue morphology and optical absorption and scattering properties, as well as local biochemistry. These signals provide detailed information regarding tissue microenvironment and disease in a non-invasive way. Because fluorescence spectroscopy can be performed with fiber optic probes, the technique is compatible with minimally invasive methods, thus allowing optical detection in internal regions of the body accessible with endoscopes or catheters. U.S. Pat. No. 6,062,591 discloses an arrangement and method for diagnosis of malignant tissue by fluorescent observation using a stroboscope as a white light source to illuminate tissues through an endoscope, while a laser stimulates the fluorescence. U.S. Pat. No. 5,377,676 discloses a method for determining the biodistribution of substances using fluorescence spectroscopy. U.S. Pat. No. 5,208,651 discloses an apparatus and method for measuring fluorescence intensities at a plurality of wavelengths.
However, while the spectrally resolved fluorescence measurements described above are relatively simple to implement clinically and appear to provide useful diagnostic information, there are certain limitations intrinsic to the steady-state spectral technique. Further, because spectrally resolved measurements are inherently intensity dependent, variations in intensity resulting from optical loss in the experimental system or optical absorption in complex media may affect the lineshape of the steady-state emission spectrum in unpredictable and unquantifiable ways. This is an important limitation to consider when applying fluorescence spectroscopy in vivo, since intensity losses attributed to hemoglobin absorption in tissue are routinely observed in vivo.
Limitations to spectrally resolved fluorescence measurements arise from the fact that these steady-state measurements integrate the emitted fluorescence signal over time, thus ignoring the dynamics of the fluorescence decay and losing an additional dimension of information. Time-resolved techniques capture the transient decay of the fluorescence intensity in time, which reflects the relative concentrations and the lifetimes of the endogenous fluorophores contributing to the emission. Because of band broadening due to molecular vibrations and non-radiant relaxation, the fluorescence emission spectrum of biomolecules is rather featureless and therefore may be relatively insensitive to local biochemical variations. Fluorophore lifetimes, which depend on both radiative and non-radiative decay mechanisms, are known to be extremely sensitive to the local biochemical environment and to vary with pH and oxygenation, both of which may differ between diseased and normal tissue. This was demonstrated explicitly for NADH, where the average lifetime was found to vary by a factor of approximately six between mitochondrial NADH and NADH in aqueous solution. Further, because fluorophore lifetime does not change with variations in excitation intensity or optical losses from hemoglobin absorption, time-resolved measurements are intensity independent.
Until recently, time-resolved fluorescence spectroscopy had been employed exclusively for in vitro diagnosis of tissue specimens, such as atherosclerotic plaque and malignant tumors. These early measurements used ultrashort laser pulses of picosecond duration for excitation and were therefore dependent upon large laser systems that would have been difficult to operate outside of the stable environment of a laser laboratory. Due to the relatively weak nature of fluorescence emission, detection was accomplished using highly sensitive photomultiplier tubes and time-correlated single-photon counting methods, which are time consuming and therefore require a static sample for measurement. For these reasons, technological limitations precluded the transfer of fluorescence lifetime spectroscopy from the laboratory to the clinic. The feasibility of using a time-resolved autofluorescence spectroscopy as a optical diagnostic technique for determining colonic polyp types in vivo, and the diagnostic accuracy of the technique was studied. (Mycek, M. A. et al., Gastrointestinal Endoscopy 1998, 48:4, 390-394)
The present invention relates to a portable Fluorescence Lifetime Spectrometer (FLS) designed to be compatible with both laboratory and clinical research studies on biological systems (cells and tissues), and which is useful to successfully discriminate cancerous and pre-cancerous tissues or cells from normal tissues or cells in vivo. The present invention also provides related methods for using the FLS to diagnose, pre-cancerous and cancerous tissues or cells and to distinguish from normal tissues or cells in vivo.